BCL keygen

Arsenic trioxide (ATO) resistance is a challenging problem in chemotherapy. However, the underlying mechanisms remain to be elucidated. In this study, we identified a high level of expression of miR-155 in a human lung adenocarcinoma A549R cell line that is highly resistant to ATO. We showed that the high level of miR-155 was associated with increased levels of cell survival, colony formation, cell migration and decreased cellular apoptosis, and this was mediated by high levels of Nrf2, NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1) and a high ratio of Bcl-2/Bax. Overexpression of the miR-155 mimic in A549R cells resulted in increased levels of colony formation and cell migration as well as reduced apoptosis along with increased Nrf2, NQO1 and HO-1. In contrast, silencing of miR-155 expression with its inhibitor in the cells, significantly decreased the cellular levels of Nrf2, NQO1 and HO-1 as well as the ratio of Bcl-2/Bax. This subsequently reduced the level of colony formation and cell migration facilitating ATO-induced apoptosis. Our results indicate that miR-155 mediated ATO resistance by upregulating the Nrf2 signaling pathway, but downregulating cellular apoptosis in lung cancer cells. Our study provides new insights into miR-155-mediated ATO resistance in lung cancer cells.

Arsenic trioxide (As₂O₃, ATO) has been successfully used in the treatment of relapsed/refractory acute promyelocytic leukemia (APL) since 1970s¹. It is also used as a treatment of solid tumors such as hepatic sarcoma, prostate, and renal cancer among others^2,3,4. It has been shown that ATO can induce cancer cell death by causing oxidative stress, DNA damage, and apoptosis⁵. Studies from our group and others have demonstrated that ATO also causes cell death in lung cancer cells^6,7 indicating that ATO may be employed for lung cancer treatment. However, the doses for ATO to induce lung cancer cell death are much higher than those for the treatment of hematologic malignancies^6,7,8, indicating that lung cancer cells are more resistant to ATO than hematologic cancer cells. Since a high dose of ATO can result in severe side effects⁹, this hinders the preclinical trials of ATO for lung cancer treatment. Thus, it is critically important to study the mechanisms underlying ATO resistance of lung cancer cells as this will help identify novel targets for attenuating ATO resistance, thereby facilitating the application of ATO as a new treatment for lung cancer.

One of the important mechanisms that underlie anticancer drug resistance is the high level and capacity of antioxidants in cancer cells¹⁰, which are primarily regulated by the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and kelch-like ECH-associated protein-1 (KEAP1) signaling pathway, one of the most important cell defense and survival pathways¹¹. Nrf2 is a critical transcription regulator of a series of antioxidants and detoxification enzymes. By uncoupling with KEAP1, Nrf2 initiates the expression of antioxidant genes including NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1)^11,12. However, previous studies have shown that cancer cells that exhibit a high level of Nrf2 are less sensitive to chemotherapeutic agents¹³. Moreover, an aberrant accumulation of Nrf2 in cancer cells confers cancer resistance to chemotherapeutic agents¹³. Because this can create an environment that promotes cancer cell growth and metastasis, but prevents cancer cells from apoptosis, thereby leading to tumor reoccurrence and poor prognosis in cancer patients¹². Our previous studies have shown that ATO significantly increases the level of Nrf2 in a human lung carcinoma cell line, A549 cell line¹⁴, suggesting that upregulation of Nrf2 is involved in resistance of A549 cells to ATO. However, the mechanism underlying Nrf2-mediated cellular resistance to ATO in lung cancer cells remains to be elucidated.

MicroRNAs (miRNAs) are a class of small non-coding RNAs (19-25 nt) that regulate protein translation and stability of mRNA¹⁵. miRNAs downregulate gene expression by binding to the 3′-untranslated region (3′-UTR) of a target mRNA, thereby inducing degradation of mRNAs and silencing the expression of a target gene¹⁵. It has been found that miRNAs play critical roles in many biological processes including cell proliferation and survival¹⁵. Dysregulation of miRNAs modulates the initiation and progression of cancer¹⁶. Moreover, a growing body of evidence indicates that several miRNAs may mediate cellular resistance to chemotherapy and radiotherapy in various types of tumors and cancer, in particular, lung cancer¹⁷. Among all of the identified miRNAs, miR-155 is the one that has been characterized extensively. miR-155 is generated from an exon of a non-coding RNA known as B-cell Integration Cluster (BIC)¹⁸. It is involved in cancer initiation and progression as well as the development of cellular resistance to chemotherapeutic agents^17,19,20,21. A previous study has shown that the level of miR-155 in lung cancer tissue is much higher than that in normal tissue²². Moreover, lung adenocarcinoma patients who exhibited a high level of miR-155 in the cancer tissue usually had poor prognosis^20,22. Inhibition of miR-155 expression suppressed cancer cell proliferation and promoted apoptosis, thereby sensitizing cancer cells to chemotherapeutic agents, cisplatin and doxorubicin^19,21. Interestingly, it has been also shown that miR-155 can upregulate NQO1 and HO-1 through activation of the Nrf2 signaling pathway, thereby protecting cells against oxidative stress^23,24. This further indicates that miR-155 can modulate cell proliferation and apoptosis via regulation of cell redox homeostasis. Our previous results have shown that a high dose of ATO can reduce the total antioxidant capacity of lung cancer cell (A549) by inhibiting cellular expression of miR-155, leading to cancer cell death (unpublished data). However, the mechanisms by which A549 cells develop ATO resistance through miR-155 remains unknown. To address this, we established a lung cancer cell line that is highly resistant to ATO, the A549R cell line, as a model to study the mechanisms by which miR-155 mediates ATO resistance in lung cancer cells. We discovered that a high level of miR-155 and activation of the Nrf2 signaling pathway along with decreased cellular apoptosis led to ATO resistance of A549R. This was further confirmed by the fact that overexpression of the miR-155 mimic enhanced ATO resistance of A549 via increased levels of NQO1 and HO-1 as well as an increased ratio of Bcl-2/Bax, whereas silencing of miR-155 with its inhibitor significantly increased the cellular sensitivity to ATO, and this resulted from decreased levels of NQO1 and HO-1 proteins as well as an decreased ratio of Bcl-2/Bax. The results indicated that miR-155 mediated cellular ATO resistance by activating the Nrf2 signaling pathway and suppressing apoptosis. Our study provided new insights into the mechanisms underlying ATO resistance in lung cancer cells. We suggest that miR-155 can be developed as a new therapeutic target for combating ATO resistance in lung cancer.

The ATO resistance of A549R cells

To determine if A549R cells exhibit a high level of resistance to ATO, we initially measured the viability of A549R cells under treatment of different doses of ATO in comparison to that of A549 cells with MTT assay. We found that the viability of A549 cells was significantly decreased by ATO that ranged from 2.5 µM to 30 µM (Fig. 1A). Treatment with 10 µM ATO for 72 hours, led to the death of more than 50% of A549 cells, whereas treatment with 20 µM–30 µM ATO resulted in the death of 80% of A549 cells (Fig. 1A). In contrast, under the same conditions, 10 µM ATO did not exhibit any cytotoxic effects on A549R cells, and 20 µM ATO only led to 20% cell death (Fig. 1A). The viability of A549R cells decreased along with increasing concentrations of ATO above 20 µM. The concentrations of ATO that caused the death of half of A549R cells fell into the range of 60 µM–80 µM, which were about 10-fold higher than that of A549 cells. The 50% inhibitory concentrations (IC₅₀) of ATO for A549 and A549R cells were 8.02 µM and 76.81 µM, respectively. Based on the IC₅₀, the drug resistance index of A549R cells was calculated as 9.58. The results indicated that A549R cells exhibited a significantly high level of resistance to ATO. To further verify the high level of resistance of A549R cells to ATO, we determined the colony formation of A549R and A549 cells without or with treatment of 20 µM ATO for 24 hours (Fig. 1B and C). Without treatment of ATO, both A549 and A549R cells exhibited a similar percentage of colony formation with 25.80% from A549 cells and 27.83% from A549R cells, respectively (Fig. 1B and C). With the treatment of 20 µM ATO, A549 cells exhibited only 3.33% of colony formation, whereas A549R cells exhibited 24.37% of colony formation (P < 0.05) (Fig. 1B and C). The results confirmed that A549R cells exhibited much higher resistance to ATO than A549 cells. We then examined the migration of A549R cells under ATO treatment to verify the drug resistant phenotype with a two-chamber system composed of an upper and lower chamber. Cell migration was measured by counting the number of cells which migrated from the upper chamber to the lower chamber (Fig. 1D and E). As shown in Fig. 1D and E, without ATO treatment, a similar number of A549 (361) and A549R cells (430) migrated from the upper chamber into lower chamber indicating that they have the similar ability of migration and survival. However, under treatment of 20 µM ATO, only 33 A549 cells migrated, whereas 421 A549R cells migrated. This indicates that A549R cells readily survived to migrate under the ATO treatment (Fig. 1E and F). Thus, the results demonstrated that A549R cells were much more resistant to ATO than A549 cells.

A549R cells exhibit poor apoptotic activity under ATO treatment

Since apoptosis can mediate ATO-induced cell death, we asked whether the high level of resistance of A549R cells to ATO was due to the modulation of apoptotic process. To address this question, we determined the percentage of apoptotic cells in untreated A549 and A549R cells as well as in the cells treated with 20 µM ATO for 24 hours and the level of apoptotic proteins in the cells. We found that lower than 0.36% of apoptotic cells were detected in untreated A549 and A549R cells (Fig. 2A, left panels and Fig. 2B). However, 20 μM ATO increased the percentage of apoptotic cells in A549 and A549R cells to 18% and 3%, respectively (Fig. 2A, right panels and Fig. 2B). Thus, A549R cells exhibited 6-fold lower percentage of apoptotic cells than A549 cells (Fig. 2A and B). This indicated that A549R cells exhibited much lower apoptotic activity than A549 cells under treatment of ATO. To further identify the molecular basis underlying the phenomena, we determined the expression levels of the pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 in the cells. We found that the level of Bax in A549R cells was lower than that in A549 cells (Fig. 2C and D), whereas the level of Bcl-2 in A549R cells was higher than that in A549 cells (Fig. 2C and D). This resulted in a lower ratio of Bax/Bcl-2 in A549R cells.

Nrf2 signaling pathway contributes to ATO resistance of A549R cells

Nrf2 mainly upregulates the expression of cellular antioxidant genes such as NQO1 and HO-1^12,13. Since our previous studies have shown that activation of Nrf2 is involved in ATO resistance in A549 cells¹⁴, it is conceivable that the Nrf2 signaling pathway may also mediate the high level of ATO resistance in A549R cells by increasing the cellular antioxidant capacity of the cells. To test this, we examined the distribution of Nrf2 in the nucleus and cytoplasm with immunofluorescence. We found that Nrf2 existed in both the nucleus and cytoplasm of A549 cells and A549R cells (Fig. 3A). However, the level of Nrf2 protein in the nucleus and cytoplasm of A549R cells was significantly higher than that in A549 cells (Fig. 3B and C). Accordingly, the levels of antioxidant proteins, HO-1 and NQO1 in A549R cells were also much higher than those in A549 cells (Fig. 3D and E). The results indicate that the Nrf2 signaling pathway was more active in A549R cells than in A549 cells.

miR-155 promotes the resistance of A549R cells to ATO

miR-155 expression is associated with increased cell proliferation and chemotherapeutic drug resistance such as cellular resistance to cisplatin and doxorubicin^19,21. Recent studies have also shown that miR-155 can upregulate the expression of NQO1 and HO-1 gene to increase cellular antioxidative capacity and cell survival^23,24. Thus, it is possible that miR-155 may mediate ATO resistance in A549R cells by upregulating NQO1 and HO-1 gene expression. To test this, we measured the level of miR-155 in A549 and A549R cells and found that the level of miR-155 in A549R cells was significantly higher than that in A549 cells (Fig. 4A). Moreover, introduction of the miRNA-155 mimic (M) or the miRNA-155 inhibitor (I) into A549R cells increased or decreased the level of miR-155 by about 2-fold compared with that of the miR-155 mimic control (MC) or the miR-155 inhibitor control (IC) (Fig. 4A). The results indicate that the miR-155 mimic or the miR-155 inhibitor can be employed to effectively regulate the level of miR-155 in A549R cells. Further characterization of the effects of miR-155 on the growth and proliferation of A549R cells under ATO treatment showed that A549R cells transfected with the miR-155 mimic exhibited a higher percentage of colony formation (29.27%) than the control (24.43%) (P < 0.05) (Fig. 4B and C), whereas A549R cells transfected with the miR-155 inhibitor exhibited a lower percentage of colony formation (15.83%) than the control (24.50%) (P < 0.05) (Fig. 4B and C). Similarly, under treatment of 20 µM ATO, 509 A549R cells transfected with the miR-155 mimic, migrated, whereas 375 A549R cells transfected with the control vector, migrated indicating that upregulation of miR-155 promoted the migration and survival of A549R cells (Fig. 4D and E). However, under the same treatment, only a small number of A549R cells transfected with the miR-155 inhibitor, migrated (Fig. 4D and E) indicating that downregulation of miR-155 suppressed cell migration and survival. The results indicate that overexpression of miR-155 promoted A549R cell survival and cellular resistance to ATO, whereas suppression of miR-155 expression inhibited A549R survival and reduced cellular resistance to ATO. This further indicated that miR-155 mediated ATO resistance of A549R cells.

miR-155 downregulates ATO-induced apoptosis in A549R cells

Since we found that ATO-induced apoptosis was significantly reduced in A549R cells (Fig. 3), it is possible that apoptosis in A549R cells may be downregulated by miR-155. To test this possibility, we determined the percentage of apoptotic cells in A549R cells that were transfected with the miR-155 mimic or the miR-155 inhibitor under treatment of 20 µM ATO for 24 hours (Fig. 5A and B). The results showed that ATO resulted in a low percentage of apoptotic cells (0.8%) in A549R cells transfected by the miR-155 mimic, and a high percentage of apoptotic cells (12.9%) in the cells transfected with the miR-155 inhibitor (Fig. 5A and B, right panels). However, ATO treatment only resulted in 3.27% and 3.13% apoptotic cells in A549R cells transfected with the vector controls of the miR-155 mimic and the miR-155 inhibitor, respectively (Fig. 5A and B, left panels). This indicated that miR-155 efficiently downregulated apoptosis. Since a low ratio of Bax/Bcl-2 can mediate anti-cancer drug resistance by preventing the initiation of apoptosis²⁵, we then asked if up- or downregulation of miR-155 could up- or downregulate apoptosis by modulating the ratio of Bax/Bcl-2. To address this, we examined the expression level of Bax and Bcl-2 proteins in A549R cells transfected with the miR-155 mimic or the miR-155 inhibitor. We found that in A549R cells transfected with the miR-155 mimic, the level of Bcl-2 and Bax was 1.46- and 0.64-fold of those in untransfected cells, respectively (Fig. 5C and D). However, the levels of Bcl-2 and Bax in A549 cells transfected with the miR-155 inhibitor was 0.71- and 1.83-fold of those in untransfected cells (Fig. 5E and F). Thus, the miR-155 mimic decreased the ratio of Bax/Bcl-2 in A549R cells to 0.44, whereas the miR-155 inhibitor elevated the Bax/Bcl-2 ratio to 2.58. The results indicate that miR-155 suppressed the initial response of A549R cells to apoptosis by decreasing the ratio of Bax/Bcl-2 through upregulating Bcl-2 and downregulating Bax.

Effects of miR-155 on the expression of Nrf2, HO-1 and NQO1 in A549R cells

Previous studies have shown that the expression of cellular antioxidants, HO-1 and NQO1can be regulated by miR-155^23,24. We have also discovered that HO-1 and NQO1 are involved in cellular ATO drug resistance²⁶. To further determine if miR-155 can mediate the resistance of A549R cells to ATO by regulating cellular levels of Nrf2, HO-1 and NQO1, we examined the levels of Nrf2, HO-1 and NQO1 in A549R cells transfected with the miR-155 mimic, the miR-155 inhibitor, the miR-155 mimic control and miR-155 inhibitor vector control, respectively. We found that the levels of Nrf2, HO-1 and NQO1 proteins were increased by about 1.5-fold in A549R cells transfected with the miR-155 mimic compared with those in the cells transfected with the mimic vector control and untransfected A549R cells (P < 0.05) (Fig. 6A and B). In contrast, the levels of HO-1 and NQO1 proteins were decreased by about 0.6-fold in A549R cells transfected with the miR-155 inhibitor compared with those in cells transfected with the inhibitor vector control (Fig. 6C and D). The results indicate that miR-155 activated the Nrf2 signaling pathway to increase the levels and capacity of antioxidants in A549R cells. This in turn enhanced the resistance of A549R cells to ATO.

In this study, we initially characterized a lung adenocarcinoma cell line, i.e. A549R cells that exhibited a significantly high level of resistance to ATO than A549 cells (Figs 1 and 2). We then explored the mechanisms underlying the ATO-resistance of A549R cells. We provided the first evidence that miR-155 mediated a high level of ATO-resistance in a lung cancer cell line, A549R cells by suppressing cellular apoptotic activities and upregulating the expression of antioxidants NQO1 and HO-1 via activation of the Nrf2 signaling pathway (Figs 2–6), and this subsequently protected A549R cells from ATO-induced apoptotic cell death, thereby promoting cell survival (Figs 4 and 5). The results support a model that miR-155 mediates the resistance of A549R cells to ATO through a pathway in which miR-155 increases the level of Nrf2 in the nucleus and cytoplasm of A549R cells. This subsequently upregulates the expression of antioxidant proteins NQO1 and HO-1 that protect cells from ATO-induced oxidative stress. On the other hand, miR-155 upregulates Bcl-2 and downregulates Bax, resulting in a high ratio of Bcl-2/Bax that subsequently prevents apoptosis. All of these promote cell survival and a high level of cellular resistance to ATO (Fig. 7).

miR-155 is implicated in the initiation and progression of cancer as well as the development of drug resistance in cancer cells^22,23. It has been shown that silencing of miR-155 leads to overexpression of the pro-apoptotic protein, Apaf-1, which increases the sensitivity of A549 cells to cisplatin²¹. Similarly, suppression of miR-155 in doxorubicin-resistant A549 cells (A549/dox) can significantly reduce doxorubicin resistance¹⁹. The results indicate that miR-155 plays a universal role in mediating anticancer drug resistance in A549 cells. In this study, we further demonstrated that miR-155 was also involved in mediating the resistance of A549R cells to ATO. Since miR-155 can upregulate NQO1 and HO-1 through activation of the Nrf2 signaling pathway to promote cellular redox homeostasis and cell survival^23,24, it appears that miR-155 may also mediate the resistance of A549R lung cancer cells to ATO by activating the Nrf2 signaling pathway and its downstream target genes. Indeed, this notion was supported by our results showing that the expression of Nrf2, NQO1 and HO-1 was upregulated along with upregulation of miR-155 in A549R cells (Figs 3 and 4A). This was further supported by the fact that overexpression of miR-155 in A549R cells increased the levels of Nrf2, NQO1 and HO-1, whereas suppression of the expression of miR-155 decreased the levels of the antioxidant proteins (Fig. 6). Thus, our results indicate that miR-155 mediated ATO resistance in A549R cells through the same cell signaling pathways as it mediates cellular resistance to other chemotherapeutic drugs. Our study further suggests that miR-155 plays a central role in mediating anticancer resistance through regulating the Nrf2 signaling pathway and can be developed as a new target for combating drug resistance of lung cancer.

Our results are consistent with previous reports showing that miR-155 plays an important role in anticancer drug resistance in cancer cells^19,21. However, a recent study has shown that miR-155 can suppress the Nrf2 signaling pathway in responding to oxidative stress induced by a different chemical compound in a different type of cells. This was demonstrated by a role of miR-155 in mediating hepatocytotoxicity induced by an environmental toxicant, perfluorooctane sulfonate (PFOS) in vitro and in vivo²⁷. In contrast to its role in mediating ATO resistance in lung cancer cells, miR-155 significantly suppresses Nrf2 expression and activation in normal hepatocytes of rats in responding to PFOS-induced oxidative stress

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Background

Emphysema is a crucial pathological characteristic of chronic obstructive pulmonary disease (COPD). Oxidative stress, apoptosis and epigenetic mechanisms contribute to the pathogenesis of emphysema. However, an attempt to accurately identify whether these mechanisms interact with each other and how they are triggered has never been conducted.

Method

The total reactive oxygen species (ROS) level, pulmonary apoptosis and B-cell lymphoma/leukemia-2 (Bcl-2) expression, an apoptosis regulator, were detected in samples from COPD patients. Bisulfite sequencing PCR (BSP) was conducted to observe the alterations in the methylation of the Bcl-2 promoter in specimens. The dysregulation of DNA methyltransferase enzyme 1 (DNMT1), a vital DNA methyltransferase enzyme, in the lungs of patients was confirmed through western blotting. To find out interactions between oxidative stress and DNA methylation in emphysema, mouse models were built with antioxidant treatment and DNMT1 silencing, and were examined with the pulmonary apoptosis, Bcl-2 and DNMT1 levels, and epigenetic alterations of Bcl-2.

Results

Higher ROS levels and pulmonary apoptosis were observed in COPD patients than in healthy controls. Downregulated Bcl-2 expression with increased promoter methylation and DNMT1 protein expression was found in COPD patients. Antioxidant treatment reduced the level of ROS, DNMT1 protein and emphysematous progression in the smoking models. Following DNMT1 blockade, smoking models showed improved lung function, pulmonary apoptosis, emphysematous progression, and increased Bcl-2 protein level with less promoter methylation than emphysema mice.

Conclusion

Cigarette-induced oxidative stress mediates pulmonary apoptosis and hypermethylation of the Bcl-2 promoter in emphysema models through DNMT1.

Chronic obstructive pulmonary disease (COPD) is a worldwide public health burden because of its high prevalence [1]. A recent study [2] showed that the estimated total number of individuals aged 20 years or older with spirometry-defined COPD in China was 99.9 million in 2015. Cigarette smoking is an established risk factor for COPD, and smoking cessation seems to be the most effective intervention for COPD [1,2,3]. However, an abundance of studies have demonstrated that pulmonary pathological progress was ongoing and could not be reversed in COPD patients after smoking cessation [4,5,6]. There are several mechanisms that contribute to the pathogenesis of emphysema, a prominent pathological hallmark of COPD, such as persistent inflammation, proteolytic/anti-proteolytic imbalance, oxidative stress, and apoptosis [4, 7, 8].

Several studies [9,10,11,12] have demonstrated that oxidative stress was a critical factor, that links inflammation, excessive matrix proteolysis and direct damage to DNA in emphysema. Our previous study and some others [13,14,15,16] showed that oxidative stress played an important role in apoptosis in COPD patients and models. Apoptosis is a highly regulated cell suicide program that can be regulated by B-cell lymphoma/leukemia-2 (Bcl-2) family proteins [17, 18]. We also demonstrated that Bcl-2 protein level was decreased in emphysema models and COPD patients, suggesting that Bcl-2 is involved in COPD pathogenesis [19]. The epigenetic regulation of DNA can stably propagate during a lifetime and might account for ongoing disease progression [20, 21]. Promoter methylation is an emerging and important epigenetic regulatory mechanism, that inhibits target gene transcription and blocks gene expression [22, 23]. Our previous studies [19, 24] found a high methylation status of the whole genome and hypermethylation of the Bcl-2 promoter in COPD patients and emphysema models, and demethylation treatment could protect models from emphysema. DNA methyltransferase 1 (DNMT1) is regarded as the primary methyltransferase, contributing to de novo DNA methylation and maintenance [25]. Based on the above studies, we postulated that cigarette smoke (CS)-induced oxidative stress mediates apoptosis and Bcl-2 methylation via DNMT1 in emphysema.

Demographic, pulmonary function and morphological findings in COPD patients

We have collected 27patients, who were underwent the excision of peripheral solitary pulmonary nodule or pulmonary lesion. Nine patients are normal nonsmokers, and 9 cases were non-COPD smokers. The last 9 cases were smokers with spirometry-defined COPD. There was no significant difference in the gender ration or age among the three groups (Table 1). The smoking history of non-COPD smokers and COPD smokers was analyzed, and no difference in smoking history was found between the two groups (P = 0.23, Table 1).

Compared with the other two groups, the COPD smokers showed destroyed pulmonary architecture and function (P < 0.05 by one-way ANOVA and least significant difference (LSD) test, Table 1 and Fig. 1a). Histologically, the nonsmokers presented a well-fixed normal pulmonary parenchyma with normal airways (Fig. 1a). Non-COPD smokers did not exhibit emphysematous changes, but presented a thicker alveolar septum than nonsmokers (mean alveolar septal thickness, MAST; Fig. 1b). COPD patients showed significant emphysematous disorder with an increased mean linear intercept (MLI) and destructive index (DI; Fig. 1c, d).

Aggravated pulmonary apoptosis and oxidative stress in the lungs of COPD patients

Compared to that in the lungs of nonsmokers and smokers without COPD, the apoptosis index (AI) was increased in the lungs of COPD patients (Fig. 2a, b). Interestingly, there was no difference in the pulmonary AI (%) between the nonsmokers and smokers without COPD (3.73 ± 1.57 vs. 3.99 ± 1.75, P < 0.01 by the Kruskal-Wallis test, Fig. 2a, b). This finding suggests that CS is not the only reason for apoptosis in COPD, and there might be mechanisms beyond direct CS damage. The level of reactive oxygen species (ROS) in tissues from COPD patients was higher than that in tissues from nonsmokers (Fig. 2c), indicating that there was a higher oxidant burden in COPD. In contrast, the smokers without COPD did not show a significant change in ROS levels (Fig. 2c).

Dysregulation of Bcl-2 and DNMT1 protein levels in lungs from COPD patients

Lung tissues from nonsmokers, non-COPD smokers and COPD patients were analyzed for the expression of Bcl-2, a well-known apoptosis regulator, by western blotting. Bcl-2 protein in lung tissue from COPD patients was lower than that in lung tissue from nonsmokers (Fig. 3a, b). To discover whether DNA methylation is involved in COPD, DNMT1 protein, the DNA methyltransferase, was analyzed in lung tissues (Fig. 3c). Immunoblotting showed that DNMT1 expression in lung tissue from non-COPD smokers and COPD patients was higher than that in lung tissue from nonsmokers (0.44 ± 0.12 and 0.73 ± 0.06 vs. 0.29 ± 0.11, P < 0.01 by one-way ANOVA and LSD test, Fig. 3c, d). In addition, the COPD group presented higher DNMT1 expression than the non-COPD smoker group (P < 0.01 by one-way ANOVA and LSD test, Fig. 3c, d).

Hypermethylation of the Bcl-2 promoter in COPD patients

Given the higher expression of DNMT1 and lower expression of Bcl-2 in COPD patients than in healthy controls, we conducted bisulfite sequencing PCR (BSP) to detect the methylation status of the Bcl-2 promoter. The sequence results demonstrated that the COPD patients had an elevated level of Bcl-2 promoter methylation (Fig. 4). As the results of Bcl-2 protein detection, BSP showed that there was no significant difference in Bcl-2 methylation levels between the nonsmoker and non-COPD smoker groups (P = 0.756, Fig. 4). Considering the simultaneously increased DNMT1 expression and methylation level, it is possible to assume that the upregulated DNMT1 level leads to the hypermethylation of the Bcl-2 promoter in COPD patients.

CS-induced oxidative stress participates in emphysema, pulmonary apoptosis and lung function damage

To confirm that oxidative stress contributes to COPD pathogenesis, we treated mice with CS and vitamin E (Vit E), one of the most common antioxidant reagents. Consistent with previous studies [13,14,15], we found that CS exposure caused increased ROS levels in lung tissue (Fig. 5a). Antioxidant feeding with Vit E prevented the increased ROS levels in CS exposed mice (Fig. 5a). Moreover, CS-treated mice exhibited emphysematous changes with aggravated MLI, DI and AI (Fig. 5b, c, d, and e). Coincidentally, the mice exposed to CS showed impaired pulmonary function, including deteriorated tidal volume (TV, mL), dynamic compliance (Cdyn, mL/cm H₂O) and airway resistance (RI, cm H₂O/mL/min; Fig. 5f, g). Interestingly, antioxidant treatment also alleviated the pulmonary morphological and functional damage caused by CS (Fig. 5b, c, d, e, f, and g). This finding suggests that CS-induced oxidative stress plays a role in emphysema, pulmonary apoptosis and lung function damage through oxidative stress.

CS-induced oxidative stress is involved in the dysregulation of Bcl-2, cleaved caspase-3 and DNMT1 protein expression and DNA hypermethylation of the Bcl-2 promoter

Because of the increased apoptosis in COPD patients’ lungs, we detected the apoptosis regulator Bcl-2 and the apoptosis inducer caspase-3 in mouse models. Compared to the control mice, the CS exposed mice had lower Bcl-2 protein expression in the pulmonary tissue (0.62 ± 0.03 vs. 0.37 ± 0.05, P < 0.01 by one-way ANOVA and LSD test, Fig. 6a, b). Correspondingly, we also found that the level of cleaved caspase-3 protein in the CS group was higher than that in control group (0.75 ± 0.09 vs. 0.15 ± 0.05, P < 0.01 by the Kruskal-Wallis test, Fig. 6a, c). Given the hypermethylation of the Bcl-2 promoter in the lungs from COPD patients, we detected DNMT1 protein expression and the DNA methylation level of Bcl-2 in pulmonary tissue from mouse models. Immunoblotting showed that DNMT1 protein level was upregulated in the lungs from the CS group (Fig. 6a, d). BSP was subsequently performed, and showed that CS led to the hypermethylation of the Bcl-2 promoter (Fig. 6e). In contrast, the antioxidant reagent ameliorated the CS-induced dysregulation of the apoptosis-associated proteins Bcl-2 and cleaved caspase-3. Moreover, it prevented the alternation in DNMT1 protein level and the CS-induced epigenetic modification of Bcl-2 (Fig. 6a, b, c, d, and e). This finding indicates that CS-induced oxidative stress causes the dysregulation of apoptosis-associated proteins and gene methylation status, and ultimately initiates apoptosis.